Micro ribonucleic acids (microRNA, miRNA or miR) are a class of non-coding, single stranded, small ribonucleic acid molecules with 19-23 nucleotides in length. They exist extensively in the cells of animals and plants and are highly conserved in evolution. MicroRNA inhibits the translation of corresponding proteins through recognizing the 3′ end non-translated sequence of target messenger RNA (mRNA) and imcompletely complementing thereto. As a powerful regulatory factor for mRNA, microRNAs are closely related to physiological activities, which relates to life activities such as the individual development, tissue differentiation, apoptosis and energy metabolism of organisms and the like; and meanwhile, microRNAs are also closely related to the occurrence and development of many diseases.
Currently, the research on plant microRNAs focuses on the regulatory effects of microRNAs on plants per se, including the effects on the growth and development, signal transduction, and behaviours under stress of plants. The application of research achievements focuses on the improvement of plant species, for example, the regulation of the expression of nutrient elements in edible parts of crops.
Patent PCT/CN 2010/000677 discloses the regulatory effects of MIR164 derived from Oryza sativa on the root system of plants, and proposes the construction of a nucleic acid fragment comprising the MIR164 sequence, which is transformed into Oryza sativa plants, thus obtaining transgenic Oryza sativa having a root system more powerful than that of general Oryza sativa. Patent PCT/IB 2010/055600 discloses that the up-regulation of several microRNAs comprising MIR156 can improve the tolerance of plants to the stress factors in environment, and then improve the biomass, vigor and yield of the plants.
Currently, the existing researches are merely limited to the regulatory effects of plant microRNAs on the physiological activities of plants per se, and the regulatory effects of plant microRNAs on the physiological activities of animals and the extraction of plant microRNAs still need investigation.
Contents of the Invention
One of the objects of the present invention is to provide a plant microRNA having the function of regulating non-plant target genes, or a plant extract containing said microRNA and preparation method and use thereof.
Another object of the present invention is to provide a method of identifying plant functional microRNAs.
In a first aspect of the present invention, provided is an isolated plant functional microRNA or an extract containing said plant functional microRNA, said plant functional microRNA is an endogenous microRNA derived from a certain plant and exists in the water-soluble and/or liposoluble extracts of said plants, moreover, said plant functional microRNA has the function of regulating non-plant target genes.
In another preferred example, said plant functional microRNAs include MIR2911; and the content of MIR2911 in said isolated plant functional microRNAs or the extract containing said plant functional microRNAs is greater than or equal to 70% by the total amount of microRNAs.
In another preferred example, the content of MIR2911 is greater than or equal to 80%; preferably, greater than or equal to 90%; and more preferably is 100%.
In another preferred example, said total amount of microRNAs refers to the sum of the amount of microRNAs of 18-24 nt in length.
In another preferred example, said regulation includes inhibition (down-regulation) of the expression of the target gene, and promotion (up-regulation) of the expression of the target gene.
In another preferred example, said non-plant target gene includes bacterial gene, viral gene, chlamydial gene, yeast gene, and animal gene.
In another preferred example, said plant includes medicinal plants, fruit and vegetable plants, and ornamental plants; preferably includes Lonicera japonica, Isatis tinctoria, Isatis indigotica, Baphicacanthus cusia, Populus diversifolia, Vigna unguiculata, cotton, Chinese cabbage or Solanum tuberosum. Preferably, said plant is Lonicera japonica, Isatis tinctoria, Isatis indigotica, Baphicacanthus cusia or Populus diversifolia; and more preferably, said plant is Lonicera japonica. 
In another preferred example, said plant functional microRNAs are microRNA species enriched in the water-soluble and/or liposoluble extracts of said plant (for example, microRNA species having an abundance of top 20, and more preferably top 10).
In another preferred example, said plant functional microRNAs further include one or more selected from the group comprising: MIR156h, MIR166f, MIR396a, MIR166a, MIR168a, MIR1440, MIR2910, MIR2915, MIR2916, MIR818d, MIR159e, MIR159c, MIR156j, MIR1432, MIR166k, MIR167b, MIR396c, MIR156e, MIR169k, MIR167c, MIR160d, MIR399a, MIR156d, MIR160e, MIR169n, MIR166l, MIR159f, MIR166c, MIR159b, MIR166j, MIR167i, MIR169c, MIR164c, MIR167j, MIR167g, MIR160c, MIR399e, MIR399b, MIR529b, MIR164e, MIR166d, MIR166h, MIR164b, MIR156f, MIR164a, MIR169l, MIR166m, MIR164f, MIR156k, MIR166g, MIR166b, MIR160b, MIR166e, MIR159d, MIR818e, MIR172a, MIR156b, MIR399g, MIR169b, MIR399f, MIR167a, MIR394, MIR156a, MIR166i, MIR167f, MIR319a, MIR156g, MIR166n, MIR399c, MIR160a, MIR159a.1, MIR156c, MIR319b, MIR169o, MIR167h, MIR156i, MIR167d, MIR169a, MIR172d, MIR818b, MIR164d, MIR167e, MIR396b, MIR2914 (Table 1).
In another preferred example, said plant extract includes the water-soluble and/or liposoluble extract of plants.
In another preferred example, said plant extract includes the extracts of branches, leaves, roots, flowers, fruits, and/or stems of plants.
In a second aspect of the present invention, provided is the use of the isolated plant functional microRNAs or an extract containing said plant functional microRNAs as described in the first aspect of the present invention in (a) preparing a composition for regulating non-plant target genes; or (b) preparing a pharmaceutical for treating diseases associated with non-plant target genes.
In another preferred example, said non-plant target gene includes bacterial gene, viral gene, chlamydial gene, yeast gene, and animal gene.
In another preferred example, said non-plant target gene is a gene of a pathogen (including bacteria, viruses, and chlamydia, etc.).
In another preferred example, said diseases associated with non-plant target genes include: tumors (such as liver cancer and lung cancer), acute and chronic infectious diseases (for example, viral diseases such as viral influenza, viral hepatitis, acquired immunodeficiency syndrome, and SARS; bacterial diseases such as tuberculosis and bacterial pneumonia; and acute and chronic infectious diseases caused by pathogenic microorganisms); and other acute and chronic diseases (for example, respiratory system diseases, immune system diseases, blood and hematopoietic system diseases such as circulatory diseases including cardiovascular and cerebrovascular diseases, metabolic diseases related to endocrine system, digestive system diseases, nervous system diseases, urinary system diseases, reproductive system diseases and motor system diseases).
In another preferred example, said plant functional microRNAs include MIR2911. More preferably, said pharmaceutical is used for treating viral influenza.
In a third aspect of the present invention, provided is a composition which comprises (a) a carrier acceptable in pharmaceutics or bromatology and (b) the isolated plant functional microRNAs and/or the plant extract containing said plant functional microRNAs as described in the first aspect of the present invention.
In another preferred example, said composition consists of or substantially consists of components (a) and (b).
In another preferred example, the content of component (b) accounts for 0.01%-99 wt % of the total weight of the composition, more preferably 0.1%-90 wt % (calculated as microRNA).
In another preferred example, said composition includes a pharmaceutical composition, a food composition or a health care product composition.
In another preferred example, the method for preparing said composition comprises the step of mixing said plant functional microRNAs or the plant extract containing said functional microRNAs with a carrier acceptable in pharmaceutics or bromatology to form said composition.
In another preferred example, said plant functional microRNAs are derived from the following plants: medicinal plants, fruit and vegetable plants, and ornamental plants, and regulates the non-plant target genes selected from the group comprising bacterial gene, viral gene, chlamydial gene, yeast gene, and animal gene.
In another preferred example, said plant functional microRNAs are derived from Lonicera japonica, Isatis tinctoria, Isatis indigotica, Baphicacanthus cusia or Populus diversifolia. More preferably, plant functional microRNAs includes MIR2911.
In a fourth aspect of the present invention, provided is a method for in vitro non-therapeutic regulation of the expression of non-plant target genes, wherein the non-plant target genes include bacterial gene, viral gene, chlamydial gene, yeast gene, and animal gene, said method comprises the step of culturing a biological material containing said target genes in the presence of the isolated plant functional microRNAs or an extract containing said plant functional microRNAs as described in the first aspect of the present invention to regulate the expression of said non-plant target genes.
In another preferred example, said target gene is a gene of a pathogen (including bacteria, viruses, and chlamydia, etc.).
In another preferred example, said biological material includes viruses, cells and tissues.
In another preferred example, said plant functional microRNAs are derived from the following plants: medicinal plants, fruit and vegetable plants, and ornamental plants.
In another preferred example, said plant functional microRNAs are derived from Lonicera japonica, Isatis tinctoria, Isatis indigotica, Baphicacanthus cusia or Populus diversifolia. More preferably, plant functional microRNAs include MIR2911.
In a fifth aspect of the present invention, provided is a method of identifying plant functional microRNAs, wherein said plant functional microRNAs have the function of regulating non-plant target genes, said method comprising the steps of:
(1) Providing an extract of a certain plant;
(2) Determining the species or levels of the plant endogenous microRNAs in said extract; and
(3) Performing alignment and analysis on the sequences of the determined plant microRNAs and non-plant target genes to identify the plant functional microRNAs having the function of regulating the non-plant target genes.
In another preferred example, said non-plant target gene includes the genes in a gene database.
In another preferred example, said non-plant target gene includes bacterial gene, viral gene, chlamydial gene, yeast gene, and animal gene.
In another preferred example, said non-plant target gene is a gene of a pathogen.
In another preferred example, said plant includes medicinal plants, and fruit and vegetable plants.
In another preferred example, microRNA species having an abundance in the extract of top 20 (more preferably top 10) are picked out in step (3) for alignment and analysis.
In another preferred example, plant microRNA species having a Lm/La ratio greater than or equal to 130% (more preferably greater than or equal to 150%; and still more preferably greater than or equal to 200%) are picked out in step (3) for alignment and analysis, wherein Lm is the abundance (or level) of a certain plant microRNA in the extract, and La is the average abundance (or level) of said plant total microRNAs.
In a sixth aspect of the present invention, provided is a plant functional microRNA molecule which is identified by the method as described in the fifth aspect of the present invention.
In another preferred example, said microRNA molecule includes MIR2911.
In a seventh aspect of the present invention, provided is a method of preparing a composition which comprises the steps of:
artificially synthesizing the plant functional microRNA molecule as described in the sixth aspect of the present invention; and
mixing said plant functional microRNA molecule with a carrier acceptable in pharmaceutics or bromatology to form the composition.
In an eighth aspect of the present invention, provided is the use of a microRNA molecule MIR2911 or an extract containing MIR2911 for preparing a pharmaceutical for treating viral influenza.
In another preferred example, said extract (unconcentrated or concentrated) contains 0.01-100 nM (preferably 0.1-20 nM) MIR2911; or
In said extract (unconcentrated or concentrated), the content of MIR2911 is greater than or equal to 70%; preferably greater than or equal to 80%; more preferably greater than or equal to 90%; and most preferably is 100% by the total amount of microRNAs.
In a ninth aspect of the present invention, provided is a method of preventing or treating diseases, wherein said diseases are diseases associated with non-plant target genes, said method comprises the step of administering the isolated plant functional microRNAs or the extract containing said plant functional microRNAs as described in the first aspect of the present invention or the composition in the third aspect of the present invention to a subject in need thereof to prevent or treat said diseases, wherein said plant functional microRNAs have the function of regulating non-plant target genes.
In another preferred example, said subject includes mammals (such as human).
In another preferred example, said non-plant target gene includes bacterial gene, viral gene, chlamydial gene, yeast gene, and animal gene.
In another preferred example, said non-plant target gene is a gene of a pathogen.
In a tenth aspect of the present invention, provided is a method of screening candidate substances of antiviral active ingredients, which comprises the steps of:
(a) Providing isolated plant functional microRNAs or an extract containing said plant functional microRNAs;
(b) Determining the stability of each microRNA in said plant functional microRNAs to select the microRNA species having high stability;
(c) Aligning said microRNAs having high stability which are selected in the above step with non-plant target genes to determine whether said microRNAs of high stability match with or bind with the non-plant target genes;
wherein, if the aligning results indicate that said microRNAs of high stability can match with or bind with the non-plant target genes, the microRNA species are selected as the candidate antiviral active ingredients.
In another preferred example, said method also comprises the steps of:
(d) Verifying whether the candidate antiviral active ingredients has an antiviral activity or not in in vitro tests or animal tests.
In another preferred example, said microRNAs of high stability are selected from the group comprising:
(i) A microRNA having the stability of top 5, preferably top 3, and more preferably top 1 among said plant functional microRNAs, or the extract containing said plant functional microRNAs;
(ii) A microRNA whose stability meets the following equationC12/C0 greater than or equal to 80%wherein C0 is the concentration when placed for 0 hour;                C12 is the concentration when placed for 12 hours.        More preferably, C0 is other than 0.        
In another preferred example, said non-plant target gene is a viral gene.
In another preferred example, in step (a), said miRNA is a species having a ranking of abundance in fresh plants (from high to low) after the fifth and preferably after the tenth.
In an eleventh aspect of the present invention, provided is a method of preparing a composition which comprises the steps of:
artificially synthesizing plant functional microRNA molecules, wherein said plant functional microRNA molecules are candidate antiviral active ingredients screened using the method described in the tenth aspect; and
mixing said plant functional microRNA molecules with a carrier acceptable in pharmaceutics or bromatology to form the composition.
In a twelfth aspect of the present invention, provided is a method of improving the abundance of MIR2911 which comprises the steps of:
(a) Providing an extract containing plant functional microRNAs, wherein said plant functional microRNAs include n microRNA species, wherein n is a positive integer greater than or equal to 2 (preferably greater than or equal to 5 or greater than or equal to 10), and wherein one microRNA is MIR2911;
(b) Subjecting said extract to aging treatment (such as placement) to obtain an aged extract and determining the abundance of MIR2911 in said aged extract, comparing the abundance with a preset value, and terminating the aging treatment when the abundance of MIR2911 is greater than or equal to the preset value to obtain an extract having an improved abundance of MIR2911.
In another preferred example, said extract is a water extract or an alcohol extract.
In another preferred example, said aging treatment is placement at 20° C.-50° C. for 2 hours to 10 days.
In another preferred example, said method also comprises using the obtained extract having an improved abundance of MIR2911 to prepare pharmaceuticals or placing same at a low temperature (such as 0° C. to −4° C. or a lower temperature) for storage.
In another preferred example, said preset value is 60%, 70%, 80%, 90%, 95%, or 99%.
In another preferred example, said abundance is calculated as the following equationabundance=S1/ΣSi×100%
wherein S1 is the amount (or concentration) of MIR2911, i=1 to n, Si is the amount (or concentration) of the ith microRNA, wherein microRNA is RNA of 18-26 nt in length.
It should be understood that all of the various technical features described above and the various technical features specifically described hereinafter (such as examples) can be combined with one another within the scope of the present invention, so as to form new or preferred technical solutions. Due to space limitations, this is no longer tired out one by one.